Isolation of spore-like cells from tissues exposed to extreme conditions

ABSTRACT

Highly undifferentiated spore-like cells can be isolated from many different tissues and bodily fluids after those tissues and fluids have been exposed to extreme conditions. The spore-like cells can be used to treat a wide variety of disorders.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation of application Ser. No. 10/020,778, filed Oct.30, 2001, now abandoned.

This application claims priority to U.S. Ser. No. 60/244,347, filed Oct.30, 2000.

TECHNICAL FIELD

The invention relates to compositions and methods for tissue engineeringand cell therapies.

BACKGROUND

Every year, millions of people suffer tissue loss or end-stage organfailure (see, e.g., Langer and Vacanti, Science 260:920–926, 1993). Whenpossible, physicians treat this loss or failure by transplanting organsfrom one individual to another, performing surgical reconstruction, orusing mechanical devices such as kidney dialyzers. Although thesetherapies have saved and improved countless lives, they are imperfectsolutions. Transplantation is severely limited by critical donorshortages, which worsen every year, and surgical reconstruction cancause long-term problems. For example, colon cancers often develop aftersurgical treatment of incontinence that directs urine into the colon.Mechanical devices are inconvenient for the patient, and theirperformance to date cannot match that of an intact organ. Few, if any,of these treatments can restore the tissue lost or prevent progressionof the underlying disorder.

An alternative to the measures described above is tissue engineering, aninterdisciplinary science that applies engineering and physiologicalprinciples to the development of biological substitutes that maintain,improve, or restore tissue function (Tissue Engineering, R. Skalak andC. F. Fox, Eds., Alan R. Liss, New York, N.Y., 1988; Nerem, Ann. Biomed.Eng. 19:529, 1991). Three general strategies have been adopted for thecreation of new tissue. The first employs isolated cells or cellsubstitutes. This approach avoids the complications of surgery, allowsreplacement of only those cells that supply the needed function, andpermits manipulation of cells before they are administered to a patient.However, the cells do not always maintain their function in therecipient, and they can evoke an immune response that results in theirdestruction. The second approach employs tissue-inducing substances. Forthis approach to succeed, appropriate signal molecules, such as growthfactors, must be purified and appropriately targeted to the affectedtissue. The third approach employs cells placed on or within matrices.In closed systems, these cells are isolated from the body by a membranethat is permeable to nutrients and wastes, but impermeable to harmfulagents such as antibodies and immune cells. Closed systems can beimplanted or used as extra-corporeal devices. In open systems,cell-containing matrices are implanted and become incorporated into thebody. The matrices are fashioned from natural materials such as collagenor from synthetic polymers. Immunological rejection may be prevented byimmunosuppressive drugs or by the use of autologous cells.

SUMMARY

The present invention is based, in part, on the discovery that highlyundifferentiated cells remain viable within, and can be isolated from,tissue that has been exposed to extreme conditions. Because these cellsare highly undifferentiated and have other unusual characteristics, someof which are reminiscent of spores, the cells are called spore-likecells. Spore-like cells are “undifferentiated” in that, when firstisolated, they have the characteristics described herein regardless ofthe tissue type from which they were isolated. The cells can lie dormantin many tissues (they have been found in all tissues examined to dateand seem to be ubiquitous), and they are also present in bodily fluidssuch as the blood. In addition, they are small, have an outer membranethat is rich in glycolipids (or glycogen) and/or mucopolysaccharides,and an unusual cytoarchitecture. They are also multipotent, and they cansurvive in extreme conditions (e.g., conditions in which differentiatedcells would die). Finally, spore-like cells fail to demonstrate activityin a microtetrazolium assay (all known living cells demonstrate redoxactivity in this assay). These characteristics are discussed in moredetail below.

Some of the methods of the invention are based on spore-like cells'remarkable ability to survive under conditions that would killdifferentiated cells or known multipotent stem cells (such as thosefound within the bone marrow, nervous system, and pancreas). Forexample, spore-like cells are tolerant of oxygen-deprivation. As shownin the Examples below, spore-like cells can survive in oxygen-poor(including essentially oxygen-free) environments, such as those thatexist within the tissues of a deceased animal (including tissues thathave been frozen for an extended period of time), or within a cappedcontainer of phosphate-buffered saline (PBS) for many hours (e.g., four,six, ten, twelve, or 24 hours or more). Differentiated or partiallydifferentiated cells (e.g., stem cells) are able to survive oxygendeprivation for variable, but much more limited, periods of time thanspore-like cells can survive the same deprivation. Differentiatedneurons are particularly sensitive, surviving in an animal for onlyabout 4–15 minutes after oxygen deprivation (caused by, for example,heart failure, cardiac or respiratory arrest, or a cerebrovascularaccident). Cartilage withstands oxygen deprivation better than mostother tissues. With refrigeration, cartilage can remain viable for amonth or so. But neither differentiated cells nor stem cells canwithstand oxygen deprivation to the same extent spore-like cells can.The spore-like cells within a tissue (or within the blood) can outlivethe differentiated or partially differentiated cells of that tissue (orwithin that blood sample) when the tissue (or blood sample) isoxygen-deprived or exposed to another of the extreme conditionsdescribed herein. Accordingly, one method of obtaining (or isolating)spore-like cells includes exposing a tissue to conditions that kill thedifferentiated or partially differentiated cells, but do not kill thespore-like cells therein. The tissue can be any biological tissue,including an intact tissue, a tissue that has been disrupted in some way(by, for example, physical dissociation), or a tissue remnant (e.g., aremnant of skin left at the scene of an accident or epithelial cellsthat have been naturally shed).

Moreover, spore-like cells can survive (i.e., remain alive after) oxygendeprivation or other harsh conditions (i.e., conditions that would killa differentiated or partially differentiated cell) without specialpreservatives. For example, hepatocytes can survive ex vivo forapproximately two days if they are specially preserved, but spore-likecells from the liver can survive for the same period (and much longer)without special preservatives.

Spore-like cells can also survive exposure to temperatures higher orlower than temperatures in which differentiated or partiallydifferentiated cells can survive. For example, spore-like cells cansurvive exposure to temperatures higher or lower than body temperature(e.g., average body temperature, elevated body temperature (as occurs,for example, with fever), or depressed body temperature (as occurs, forexample, with hypothermia)). For example, spore-like cells remain viablewithin (and can be isolated from) tissues that are stored at about 4° C.for a prolonged period of time (e.g., one, three, five, seven, or moredays). They also remain viable at temperatures that vary even furtherfrom a physiological body temperature. For example, substantially purepopulations of spore-like cells (e.g., spore-like cells isolated from amammal) and spore-like cells within tissues (e.g., spore-like cellswithin mammalian tissues) can survive freezing or heating to more than5° C. in excess of a physiological body temperature. That is, viablespore-like cells survive (e.g., within tissues) exposure to temperaturesof 0° C., or below, or 43° C. or above (e.g., 45, 50, 55, 58, 60, 75,90, 95, or 100° C.). As with oxygen-deprivation, spore-like cells cansurvive exposure to these conditions without special treatment (e.g.,they can survive exposure to freezing temperatures even withouttreatment with a cryopreservative). Viable spore-like cells can also beisolated from tissues that have been thoroughly dried (e.g., byplacement in a dessicator for approximately 4, 8, 12, or 24 hours ormore).

Because spore-like cells can survive exposure to extreme conditions,they can be isolated from tissues that have been exposed to any one ofthose conditions (the differentiated or partially differentiated cellshaving been destroyed by the condition). For example, spore-like cellscan be isolated from an animal (including a human) that has been deadfor many hours, for several days, or longer. The precise time is notcritical. What is important is that the conditions be such thatspore-like cells in the tissue of interest survive after thedifferentiated or partially differentiated cells have died (that is notto say that every spore-like cell that was in the living animal ortissue sample need survive; any number of spore-like cells can survivewhere no differentiated or partially differentiated cells survive). Asindicated above, differentiated cells within oxygen-sensitive tissues,such as the brain, are not viable after only short periods of oxygendeprivation. Thus, the precise conditions required vary depending on thetissue type used for the isolation procedure; the extent of thetreatment (e.g., the time the tissue must be oxygen-deprived) need onlybe sufficient to kill the differentiated or partially differentiatedcells in the tissue used.

Spore-like cells can also be isolated from tissues that have been frozenwithout a cryopreservative. Thus, spore-like cells can be isolated fromany tissue (be it intact or processed or manipulated in some way) thathas been placed in a freezer without first being treated with acryopreservative (as noted above, the time period of exposure need onlybe sufficient to kill the differentiated or partially differentiatedcells in the animal, tissue, or bodily fluid). Similarly, spore-likecells can be isolated from animals that have died in the wild in frigidclimates and, quite probably, from animals that have been frozen formany, many years. Similarly, because spore-like cells remain viable evenafter exposure to heat, they can also be recovered from animals thathave died in fires, arid landscapes, or in warm springs. Some of theanimals from which spore-like cells can be isolated may now be extinct.

Although spore-like cells can be isolated from tissue that has beenfrozen (or boiled), methods of isolating spore-like cells can be carriedout at temperatures above freezing (or below boiling). Here again, thetemperature must only be low enough or high enough to killdifferentiated or partially differentiated cells.

Spore-like cells can be used to analyze fundamental aspects of cellulardifferentiation and to treat many types of disorders. For example,spore-like cells can be used to reengineer damaged or diseased tissue,to augment existing tissue, to create new tissue, or to otherwiseimprove the condition of a patient who is suffering from a disorder thatis amenable to treatment by a cell- or gene-based therapy. For example,spore-like cells that differentiate into various types of skin cells canbe used to repair skin damaged by physical, thermal, or chemical trauma.Similarly, spore-like cells that differentiate into insulin-secretingcells can be used to treat diabetes; spore-like cells that differentiateinto α-galactosidase A-expressing cells can be used to treat Fabrydisease; and spore-like cells that differentiate into cells that expressangiogenesis inhibiting factors, such as an endostatin, or otheranti-tumor agents (e.g., tumor necrosis factor), can be used to treatcancer. These are merely examples of the ways in which spore-like cellscan be used. Alternatively, or in addition, one can use spore-like cellsthat are engineered to secrete substances such as those described above.The cells can be made to express a wide variety of substances by geneticmanipulation or exposure to factors that alter their course ofdifferentiation. Spore-like cells can also be used to treat patients whohave an infection. It is believed that spore-like cells are so primitivethat they remain unaltered by exposure to agents that infectdifferentiated cells. For example, a patient who has hepatitis can betreated by harvesting a portion of the liver, isolating spore-like cellsfrom that tissue sample, and using the spore-like cells to reengineermature liver cells and tissues. All or a portion of the infected livercan be ablated before the reengineering process. Similarly, one cantreat patients who have cancer using spore-like cells. For example,spore-like cells can be isolated from a patient who has a type ofleukemia before the patient undergoes chemotherapy or any other therapyfor the cancer. Following therapy, spore-like cells, which may have beeninduced to differentiate ex vivo, can be administered to the patient toreconstitute a healthy cadre of blood cells. There is no evidence thatspore-like cells are susceptible to the processes that result inmalignancy, and the therapy administered can be very aggressive (andthereby more likely to kill malignant blood cells and thus, succeed ineradicating the cancer). The methods of the present invention may alsobe useful in forensic science. For example, one can isolate spore-likecells from a deceased person or from a sample (e.g., a blood sample)found at a crime scene and use the cells to identify the person that wastheir source. For example, one can place the spore-like cells inculture, allow them to differentiate, and analyze their genetic materialby standard techniques.

As noted above, spore-like cells can be isolated from a tissue,including a tissue that has been exposed to any condition that isextreme enough to kill the differentiated or partially differentiatedcells within the tissue. For example, the tissue can be exposed to anoxygen deficient environment, a non-physiological temperature, aninsufficiently moist environment (as in, for example, a dessicator), ora toxin or any other substance (e.g., a salt or improperly bufferedsolution) or event (e.g., radiation) that kills differentiated orpartially differentiated cells. In particular embodiments, spore-likecells can be isolated from a tissue that was harvested from an animalwhose heart ceased beating at least four minutes ago (i.e., an animalthat has been dead for at least 4, 10, 20, or 30 minutes; at least 1, 2,4, 10 or 24 hours; at least 2, 4, 7, 10 or 30 days; at least 5, 10, 20or 40 weeks; or at least 1, 2, 4, 10 or 100 years). Generally,differentiated cells will not survive unless they are within 200 μm ofan oxygen supply (such as a blood vessel carrying oxygenated blood), butspore-like cells can survive even if they are more than 200 μum awayfrom such an oxygen supply.

In other embodiments, spore-like cells can be isolated from a tissuethat was (or has been) exposed (e.g., placed in a bath of hot or coldwater, a cold room, freezer, or the like) to a temperature that is morethan 42° C. or less than 0° C. without first being treated with aprotective agent (e.g. a cryopreservative such as glycerol). Theisolation procedure following exposure to any extreme condition can becarried out very simply. For example, a biological sample that containscells such as tissue cells or a bodily fluid that has been exposed to acondition extreme enough to kill differentiated or partiallydifferentiated cells can simply be placed in a tissue culture vessel(e.g., a plate or flask). The dead cells can then be washed away afterthe spore-like cells adhere to the vessel. If desired, the tissue can bedisrupted (by, e.g., cutting, shredding, or scraping it with a bluntinstrument) either before or after it is placed in culture or before orafter exposure to an extreme condition. Given spore-like cells' abilityto survive in extreme conditions, there is no reason to expect that theywould not survive in culture under most, if not all, of the conditionsused to culture differentiated cells. Alternatively, or in addition,spore-like cells can be isolated by passing a tissue, bodily fluid, orcell culture medium that contains them through a series of devices(e.g., size-exclusion devices such as pipettes or filters) havingprogressively smaller apertures (the smallest of which can beapproximately 15 μ). Smaller diameters (i.e., diameters smaller than 15μ) can also be used when more aggressive isolation is desired (i.e.,when one desires fewer differentiated cells in the resulting culture).More aggressive isolation may be desired when one wishes to maintain thespore-like cells in their highly undifferentiated state. As describedbelow, the conditions in which the cells are cultured can be such thattheir proliferation is encouraged and their differentiation isdiscouraged.

The ability to survive after being exposed to a condition that killsdifferentiated or partially differentiated cells is only one of theunusual characteristics that can be exploited to identify and isolatespore-like cells. They can also be identified and isolated on the basisof their size alone. Although there is some variation in the size ofspore-like cells (see below), it is clear that many spore-like cells aresmaller than any other known biological cell. Accordingly, one canisolate spore-like cells by isolating the smallest cells in a tissue orbodily fluid. The isolation can be carried out by any method known inthe art (e.g., flow cytometry). Alternatively, the method can be carriedout using a size-exclusion device, such as the pipettes and filtersdescribed above.

In another aspect, the invention features isolated cells that arenon-terminally differentiated progeny of spore-like cells that wereisolated from non-neural and non-pancreatic tissues and that developinto mature non-neural and non-pancreatic cells.

One way to distinguish the cells of the present invention frompreviously described cell types is to isolate the present cells fromtissues where no stem cells are known to exist. For example,conventional wisdom dictates that there are no stem cells in the liveror the heart. Therefore, any cell isolated from the liver or heart thatis a dividing, non-mature cell is a spore-like cell of the presentinvention or a non-terminally differentiated progeny thereof.

Spore-like cells can be administered alone, with other cell types, or inconjunction with tissue engineering constructs (i.e.,tissue thatincludes materials or devices used to reengineer damaged, diseased, orotherwise unhealthy tissue). These constructs can include supportstructures, such as a mesh, or a hydrogel. Together, the hydrogel andthe spore-like cells of the invention form a hydrogel-spore-like cellcomposition. Similarly, a hydrogel combined with a progenitor cell formsa hydrogel-progenitor cell composition. Thus, the invention featuresmethods for generating an artificial tissue by, for example, combininghydrogel with a spore-like cell or the progeny of a spore-like cell. Thehydrogel-cell compositions can also be delivered into a permeable,biocompatible support structure and used to treat damaged tissue (e.g.,a hydrogel-spore-like cell composition can be applied to the damagedtissue).

In addition to methods for generating and repairing tissue, theinvention features methods of treating patients who have a disorder,such as a skin disorder, a tumor, or a disease, such as diabetes. Thesemethods are carried out, for example, by administering a spore-like cellor its progeny to the damaged region (e.g., the damaged region of thepatient's skin, the area from which the tumor was ablated, or thepancreas). Systemic administration is also possible. The methods of theinvention can be used to treat a patient who has a deficiency offunctional cells in any of a wide variety of tissues or systems,including the retina (or other structures associated with vision),auditory system, nasal epithelium, alimentary canal, pancreas,gallbladder, bladder, kidney, liver, heart, lung (including respiratorysupport structures such as the trachea and smaller airways), nervoussystem, reproductive system, endocrine system, immune system, bone,muscle, tooth, nail, or skin (including the hair follicles).

Spore-like cells and their progeny must originally be isolated fromtheir natural environment (i.e., removed from a place where they residewithin an animal) to fall within the present invention. Spore-like cellscan be isolated from tissues that are derived from the endoderm,mesoderm, or ectoderm. Similarly, spore-like cells can differentiateinto tissues that derive from the endoerm, mesoderm, or ectoderm. Thegerm layers, and the tissues they give rise to, are well known in theart. An “isolated” spore-like cell can be one that is placed in cellculture, even temporarily. The term covers single, isolated spore-likecells and their progeny, as well as cultures of spore-like cells (andtheir progeny) that have been significantly enriched (i.e., cultures inwhich less than about 10% of the cells are differentiated or partiallydifferentiated cells).

The term “disorder” encompasses medical disorders, conditions,syndromes, illnesses, and diseases, regardless of their etiology. Forexample, a disorder amenable to treatment with the compositions andmethods described herein can be caused by trauma, a genetic defect, aninfection, substance abuse, uncontrolled cellular proliferation, or adegenerative process (e.g., neural degeneration or muscular atrophy). Agiven disorder is treated when the symptoms of the disorder arealleviated or the underlying cause is eliminated or counteracted, eithercompletely or partially.

A “hydrogel” is a substance formed when an organic polymer, which can benatural or synthetic, is set or solidified to create athree-dimensional, open-lattice structure that entraps molecules ofwater or other solutions to form a gel. Solidification can occur byaggregation, coagulation, hydrophobic interactions, cross-linking, orsimilar means. Preferably, the hydrogels used in conjunction withspore-like cells solidify so rapidly that the majority of the spore-likecells are retained at the application site. This retention enhances newcell growth at the application site. However, those of ordinary skill inthe art will recognize that cellular retention is not always necessary.For example, retention is not necessary when treating a systemicdisorder. The hydrogels are also biocompatible (e.g., they are not toxicto cells). The “hydrogel-cell composition” referred to herein is asuspension that includes a hydrogel and a spore-like cell or itsprogeny.

There are many advantages to using spore-like cells. For example, theycan be used to produce sufficient biological material for tissueengineering. This is not always possible when fully differentiated cellsare used as the starting material. In addition, spore-like cells candifferentiate into a greater variety of cell types than previouslyidentified progenitor cells isolated from adult mammals.Thus,.spore-like cells can be used to maintain or repair many, if notall, tissues and organs, including those (such as the retina) that havenot been considered likely candidates for tissue engineering. Thepluripotent nature of spore-like cells also allows more histologicallycomplete development of any given tissue. For example, spore-like cellscan be used to engineer skin that is pigmented and that contains adnexalstructures (i.e., accessory structures or appendages such as hairfollicles, sweat glands, sebaceous glands, nail beds, and specializedsensory receptors that allow us to sense pain, pressure, temperature,position, etc). The pigmentation and adnexal structures render the skinreplacement a more visually appealing and functional replacement fornatural, undamaged skin. Of course, disorders affecting the skin areonly one of the many types of disorders that can be treated withspore-like cells. Analogous benefits will be apparent when systemicdisorders or disorders affecting other organs (e.g., the pancreas,liver, heart or lung) are treated. The use of spore-like cells may alsoobviate the need to obtain cells or tissues from embryonic or fetaltissue and may therefore diffuse the emotional and political debate thatcurrently surrounds the research and treatments that rely on embryonicor fetal tissue.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, useful methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflicting subject matter, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A–1C are scanning electron micrographs of spore-like cellsobtained from the liver of an adult rat. The cells are magnified 5,000×in FIGS. 1A and 1B, and 10,000× in FIG. 1C. The scale bars represent 1.0μ.

FIGS. 2A–2D are transmission electron micrographs of spore-like cellsobtained from the liver of an adult rat and placed in culture for 12days. The magnification in FIGS. 2A–2D is 25,000×, 39,000×, 17,000×, and90,000× respectively.

FIGS. 3A–3C are photographs of cells isolated from an adult rat heartand placed in culture. The newly isolated cells shown in FIG. 3A includeundifferentiated spore-like cells (magnified 100×). After three days inculture, early myocardial cells can be seen (FIG. 3B). After two weeksin culture, Purkinje-like structures can be seen (FIG. 3C).

FIGS. 4A–4C are photographs of cells isolated from the small intestineof an adult rat. The newly isolated cells shown in FIG. 4A includeundifferentiated spore-like cells. After three days in culture, clustersof small intestinal cells (FIG. 4B) and autonomic neurons (FIG. 4C) canbe seen. FIGS. 4A–4C are shown at a magnification of 200×.

FIGS. 5A and 5B are photographs of cells isolated from the bladder of anadult rat. The newly isolated cells shown in FIG. 5A includeundifferentiated spore-like cells (magnification at 100×). After twodays in culture, the isolated spore-like cells, or their progeny, appearto be differentiating (FIG. 5B; magnification at 200×).

FIGS. 6A and 6B are photographs of cells isolated from the kidney of anadult rat. The newly isolated cells shown in FIG. 6A includeundifferentiated spore-like cells (magnification at 100×). After threedays in culture, aggregates of cells resembling kidney structures can be(FIG. 6B; magnification at 200×).

FIGS. 7A–7E are photographs of cells isolated from the liver of an adultrat. The newly isolated cells shown in FIGS. 7A and 7C includeundifferentiated spore-like cells (magnification at 100×). After threedays in culture, an aggregate of cells resembling a differentiatingliver structure can be seen (FIG. 7B; magnification at 200×). Afterseven days in culture, cells resembling hepatocytes can be seen (FIG.7D). After 12 days in culture, many cells isolated from the liverexpress bile, as evidenced by a Hall's stain (FIG. 7E; 400×).

FIGS. 8A–8C are photographs of cells isolated from the lung of an adultrat; FIG. 8D is a photograph of cells in a culture initiated byspore-like cells obtained from an adult sheep lung; and FIG. 8E is aphotograph of a semi-thin section of a feline lung. The newly isolatedcells shown in FIG. 8A include undifferentiated spore-like cells. Aftersix weeks in culture, alveolar-like cells can be seen (FIGS. 8B and 8C).After 30 days in culture, spore-like cells have formed alveolar-likestructures (FIG. 8D) similar to those seen in the lungs of adult mammals(FIG. 8E).

FIGS. 9A–9D are photographs of cells isolated from the adrenal-gland ofan adult rat. Undifferentiated spore-like cells can be seen at Day 0(see the arrows in FIGS. 9A (200×) and 9B (400×)). After two days inculture, primitive adrenal cells can be seen (FIGS. 9C (200×) and 9D(400×)).

FIGS. 10A–10C are photographs of islet-like structures. These structuresformed in cultures of spore-like cells that were isolated frompancreatic tissue that contained no islets (the islets were harvestedprior to the isolation of spore-like cells). After six days in culture,more than 100 islet-like structures were present per field (at 100×magnification; FIGS. 10A and 10B). The islet-like structures wereimmunostained, which revealed insulin expression (FIG. 10C).

FIG. 11 is a photograph of a culture that includes undifferentiatedspore-like cells isolated from adult human blood.

FIGS. 12A and 12B are photographs of cultured cells. The cultures wereestablished seven days earlier and, as shown by phase contrastmicroscopy, contained spore-like cells isolated from adult human blood(FIG. 12A). Immunofluorescent staining was then performed and, some ofthe cells expressed nestin (FIG. 12B).

FIG. 13 is a schematic of a permeable support structure filled with ahydrogel-spore-like cell composition.

DETAILED DESCRIPTION

The present invention provides compositions and methods for repairing,replacing, or generating tissue or another biologically useful substance(e.g., a hormone, an enzyme, an anti-angiogenic factor, a cytokine, agrowth factor, or other biologically active molecules, includingchimeric molecules (e.g., a polypeptide that contains a detectablemarker)). The compositions include spore-like cells (e.g., mammalianspore-like cells), and can be used to study cellular differentiation, todetect a compound that has an adverse effect on differentiation (andtherefore a possible adverse effect on development) or administered to apatient either by the methods described below or by way of existingtissue engineering or cell therapy procedures known to those of ordinaryskill in the art. For example, to screen compounds for possible adverseeffects on development, one can expose spore-like cells to thecompound(s) and follow their course of differentiation. If the compoundinhibits or alters cellular differentiation (relative to an appropriatecontrol, such as a spore-like cell or population of spore-like cellsthat have been similarly treated but not exposed to the compound), thenthe compound may have an adverse effect on development (e.g., fetaldevelopment) and should be tested further.

When spore-like cells are fused in cell therapies, they can beadministered just as more differentiated cells have been administered.For example, when spore-like cells are used to treat diabetes, they canbe administered just as mature insulin-expressing cells have beenadministered (e.g., by implantation under the renal capsule or withinvarious implantable or extracorporeal devices). Spore-like cells canalso be placed within a containment device and implanted, for example,within a patient's abdomen to treat a variety of disorders. This methodof administration is particularly well suited for treating systemicdisorders, such as those caused by an enzymatic imbalance. Implantationby way of containment devices is also useful when cells requireprotection from the patient's immune system (however, a particularadvantage of administering spore-like cells is that they do not requiresuch protection—they may be harvested from the patient to whom they aresubsequently administered or they may be so primitive that they fail toevoke an immune response).

Alternatively, as described below, spore-like cells can be combined witha liquid hydrogel that can be placed into a permeable, biocompatiblesupport structure that is delivered to a patient (either before or afterit is filled with the hydrogel-cell composition). As the hydrogel-cellcomposition fills the support structure, it assumes the structure'sshape. When spore-like cells proliferate and differentiate to such anextent that they form new tissue, the support structure guides the shapeof the developing tissue. For example, the support structure can beshaped as a bone (or a fragment thereof), a meniscus within a joint, anear, an internal organ (or a portion thereof), or other tissue (e.g.,the skin). However, the support structure need not be strictly fashionedafter naturally occurring tissue in every case. For example, the supportstructure can be shaped in a way that simply facilitates delivery ofspore-like cells to a patient. For example, the support structure can beshaped to fit under the renal capsule or within some other organ orcavity (e.g. the support structure can be shaped to lie within a portionof the gastrointestinal tract or to fill a space once occupied bytissue, such as the spaces created when a tumor is surgically removed orwhen a tissue has been destroyed following trauma, ischemia, or anautoimmune response).

In some instances, including instances where spore-like cells areadministered in the course of cell or gene therapy, spore-like cells canbe administered without containment devices, hydrogels, or supportstructures. It is well within the ability of one of ordinary skill inthe art to determine when spore-like cells should be confined within aspace dictated by a support structure and when they should not. Forexample, one of ordinary skill in the art would recognize that whentreating respiratory distress syndrome (RDS) with spore-like cells thatare made to secrete surfactant, or that differentiate into cells thatsecrete surfactant, the substance that reduces surface tension withinthe alveoli, they must be supplied locally.

Spore-like cells, and their progeny, and exemplary methods for theirisolation and administration, are described below.

Spore-Like Cells

As noted above, spore-like cells were so named because they havecharacteristics reminiscent of those of spores. More specifically,spore-like cells have one-or more of the following characteristics.First, they are widely distributed throughout the body. They have beenisolated from every tissue examined to date, and they have been isolatedfrom bodily fluids as well. Thus, they can be isolated from a widevariety of tissues (e.g., cardiac, smooth and skeletal muscle,intestine, bladder, kidney, liver, lung, adrenal gland, skin, retina,nasal epithelium, brain, spinal cord, periosteum, perichondrium, fascia,and pancreas) and bodily fluids (e.g., blood, cerebrospinal fluid,urine, and saliva). Stated more generally, spore-like cells can beisolated from tissues that are generated from the endoderm, mesoderm, orectoderm.

Second, spore-like cells are multipotent, i.e., they can differentiateinto two or more (e.g., two, three, four, five, or more) cell types. Forexample, multipotent spore-like cells can differentiate into epithelialcell, keratinocytes, and melanocytes. They can also self-replicate. Thatis, a spore-like cell can divide to produce either one or two newspore-like cells. Spore-like cells can differentiate into the cell typesof the tissue from which they were isolated (e.g., spore-like cellsisolated from the pancreas can differentiate into insulin-producingislet cells or glucagon-producing islet cells) or into the cell types ofanother tissue. For example, spore-like cells isolated from the bloodcan differentiate into insulin-producing islet cells, and spore-likecells isolated from cartilage or periosteum can differentiate intoneurons.

Third, spore-like cells are small. Most spore-like cells have a diameterof approximately one to seven microns (e.g., a diameter of one to two,two to four, three to five, about five, or five to ten microns).However, spore-like cells having a diameter of less than approximatelyone micron in diameter (e.g., one-tenth, one-fifth, one-third, orone-half of a micron) have also been observed). The diameter of thespore-like cells may increase somewhat as they adhere to a tissueculture vessel and “sit down.” Cell size is discussed further below.

Fourth, spore-like cells appear to be membrane-bound, as biologicalcells typically are, but within the membrane or associated with themembrane, spore-like cells have an unusually high content of glycolipids(or glycogen) and/or mucopolysaccharides. In fact, there are sufficientglycolipids (or glycogen) and/or mucopolysaccharides that the cellsappear to have one or more dark stripes when viewed under a powerfulmicroscope (by, e.g., transmission electron microscopy). When the cellsare exceedingly small (e.g., less than about one or two microns), thestripes are not as obvious, but they can, even in some of theexceedingly small cells, be seen with a trained eye. While it is notpresently known, glycolipids (or glycogen) and/or mucopolysaccharidesmay help protect spore-like cells and thus better equip them to surviveharsh conditions, such as those described herein.

Fifth, spore-like cells have-an unusual cytoarchitecture. Electronmicrographs and histological stains for nucleic acids reveal that alarge portion (e.g., at least about 50% and up to about 90% or more) ofthe volume of a spore-like cell is comprised of nucleic acids.Mitochondria have also been observed.

Sixth, spore-like cells can survive in extreme conditions (i.e.,conditions in which differentiated or partially differentiated cellswould die). For example, spore-like cells are tolerant ofoxygen-deprivation. As shown in the Examples below, spore-like cells cansurvive in low-oxygen environments, such as those that exist within thetissues of a deceased animal, for many hours (e.g., four, six, ten,twelve, or 24 hours or more).

Spore-like cells can be obtained by the methods of the present inventionfrom many different types of donors (e.g., a member of an avian,reptilian, amphibian, or mammalian class). For example, mammalianspore-like cells can be isolated from a rodent, a rabbit, a cow, a pig,a horse, a goat, a sheep, a dog, a cat, a non-human primate, or,preferably, a human. Spore-like cells can be obtained from an animaleven after it has reached adulthood. Because spore-like cells tolerateoxygen deprivation and exposure to extreme temperatures better thandifferentiated cells, viable spore-like cells can also be isolated fromdeceased animals, including animals that have been deceased for manydays, if not weeks, months, or years (e.g., animals that have beendeceased for 1,000 years or more).

In addition, spore-like cells can be obtained from a variety of sourceswithin a given donor. For example, spore-like cells can be obtained frombodily fluids (e.g., blood, saliva, cerebrospinal fluid, or urine), andmost, if not all, functional organs and mucous membranes. Moreover,spore-like cells can be obtained from the patient who will besubsequently treated with those cells, from another person, or from ananimal of a different species. In other words, autologous, allogenic,and xenogeneic spore-like cells can be obtained and used to treat humanpatients.

Regardless of the source from which they are obtained, spore-like cellscan be placed in culture, and cell lines derived from spore-like cellscan be developed. Given the unique characteristics of spore-like cells,it is entirely reasonable to think they are immortal (i.e., they can becultured indefinitely). However, if this is not the case, they can beimmortalized using techniques routinely practiced by those of ordinaryskill in the art. Thus, cultured spore-like cells and cell lines derivedfrom spore-like cells can also be used to treat human patients.

Spore-like cells can differentiate into many different cell types. Forexample, as shown below, spore-like cells can be isolated from adultmammalian liver, lung, heart, bladder, kidney, and intestine, and candifferentiate into hepatocytes, alveolar cells, cardiac myocytes,bladder cells, renal cells, and autonomic neurons, respectively.Spore-like cells obtained from the retina can differentiate into cellshaving morphologies similar to that of rods and cones. Spore-like cellsobtained from the spinal cord can differentiate into cells havingmorphologies similar to that of neurons, astrocytes andoligodendrocytes.

Spore-like cells can also be isolated from readily obtainable bodilyfluids, such as the blood. Given the variety of known sources forspore-like cells, it is reasonable to expect that these cells can befound in most, if not all, tissues and bodily fluids. Similarly, giventhe number of differentiated phenotypes already observed, it isreasonable to expect that spore-like cells can differentiate into most,if not all, types of cells.

As noted above, spore-like cells are generally spherical, especiallywhen first isolated, and are typically small. Many cells in a culture ofnewly isolated spore-like cells are approximately 1 to 3 μ in diameter.However, larger and smaller spore-like cells have been identified (e.g.,using electron microscopy; see Example 2). Given that spore-like cellscan differentiate into a variety of mature cell types, and thatdifferentiation is a gradual process, it is difficult to define theprecise upper size limit of spore-like cells. However, spore-like cells4 to 5, as well as 7 to 10, μ in diameter have been identified inscanning electron micrographs. Occasionally, even larger cells (e.g.,cells as large as 12 to 18 μ or more) have been observed. The largercells may have in fact been cells on the verge of cell division(structures resembling mitotic clefts are often visible). Alternatively,the larger cells may really be conglomerates of several spore-likecells.

The lower size limit of the spore-like cells is more definite and iscertainly unique. Spore-like cells that are only about one-third of amicron in diameter have been observed in scanning electron micrographsand some cells may be as small as one-tenth of a micron. This extremelysmall size may reflect the unique composition of spore-like cells. Newlyisolated spore-like cells contain a great deal of nuclear material andrelatively little cytoplasm. In most differentiated cells, the nucleusconsumes approximately 10–20% of the cells' volume. However,approximately 50% and up to approximately 90% of the volume of aspore-like cell is consumed with nuclear material. The nuclear materialappears to be surrounded by a coat containing glycolipids and/ormucopolysaccharides.

Without limiting the invention to spore-like cells that arise by anyparticular mechanism, it is believed that spore-like cells may arisewhen essential DNA fragments (which may represent compressed DNA) areshed from mature cells (e.g., those undergoing cell death by apoptosisor other means) and re-packaged in a glycolipid-rich coat. Indeed, theconcept of a minimal genome is beginning to emerge. This concept isexemplified by a mycoplasma that contains 517 genes but only requires265 to 350 of these genes to survive (Hutchison et al. Science286:2165–2169, 1999). If one considers the exquisite simplicity of DNAand the genetic code, it seems plausible that the complex informationstored in DNA could be compressed considerably.

The unique size and composition of newly-isolated spore-like cell isperhaps best appreciated by viewing the cells with an electronmicroscope (e.g., see FIGS. 1A–1C and 2A–2D).

Functionally, spore-like cells are unique in at least three ways. First,even though they are present in mature animals (e.g., post-natal,adolescent, or adult animals), they can differentiate into a widevariety of different cell types. Second, spore-like cells tolerate(i.e., survive following exposure to) conditions that killdifferentiated or partially differentiated cells (e.g.,oxygen-deprivation and exposure to temperatures that are either muchhigher or much lower than normal body temperature (which, forwarm-blooded mammals, is 37° C.)). Experiments have demonstrated thatspore-like cells can tolerate essentially complete oxygen deprivationfor at least five days (cells were viable despite oxygen deprivation foreither four hours, 24 hours, or five days). Thus, spore-like cells cantolerate prolonged oxygen deprivation for at least five days or longer.In addition, spore-like cells have a greater capacity to proliferatethan terminally differentiated cells isolated from specialized tissues.Proliferative capacity is an important attribute because tissueengineering, cell therapies, and gene-based therapies are often hamperedby physicians' inability to obtain sufficient numbers of cells toadminister to a patient.

To obtain spore-like cells, a sample is obtained from an animal, such asa human. One of the easiest samples to obtain is a sample of wholeblood. Those of ordinary skill in the art will appreciate that theisolation method may vary slightly depending on the type of tissue usedas the starting material. For example, in the event the sample is ablood sample, it can be placed in a tube containing an anti-coagulant.After collection, tissue samples, whether they are samples of bodilyfluids, organs, tissues, or cell suspensions thereof, can be stored.Moreover, spore-like cells can be recovered from bodily fluids or othersamples that have been stored under conditions in which differentiatedcells or known stem cells (e.g., hematopoietic stem cells) cannotsurvive (e.g., frozen storage without a cryopreservative).

Either immediately after collection or after storage (e.g., under normalcell storage conditions, but also in an oxygen-poor environment or at atemperature more than 42° C. or at or below freezing), the cells can becentrifuged for a time and at a speed sufficient to pellet them in thebottom of the centrifuge tube. The resulting pellet is resuspended in asuitable medium (e.g., DMEM/F-12 medium supplemented with glucose,transferrin, insulin, putricine, selenium, progesterone, epidermalgrowth factor (EGF) and basic fibroblast growth factor (bFGF; see theExamples, below. Other media have been used and have worked as well asthat just described for the growth, proliferation, and differentiationof spore-like cells. After collection, the tissue sample can beintentionally exposed to harsh conditions, for example, those describedherein, to kill differentiated cells.

The suspended cells are then transferred to a tissue culture vessel andincubated (obviously, the incubation temperature is not critical, butmost incubators are kept at or near 37° C.). Initially, when the sampleis a blood sample, the culture flasks contain primarily hematopoieticcells. However, after several days in culture, the red blood cells lyseand degenerate so that the culture contains primarily, if notexclusively, spore-like cells. When spore-like cells are isolated fromsolid tissues, the differentiated cells can be lysed by triturating thesample with a series of pipettes, each having a smaller bore diameterthan the one before. For example, the last pipette used can have a borediameter of approximately 15 μ (methods in which spore-like cells areisolated after exposure to an extreme condition and methods in whichspore-like cells are isolated based on another characteristic, such assize, or their ability to withstand infectious agents, can be carriedout separately or in combination). After several additional days inculture, the spore-like cells multiply and can coalesce to form clustersof cells. Over time, usually on the order of approximately 7 days, theirnumber can increase greatly. Typically, more than 90% of the spore-likecells are viable according to Trypan blue exclusion studies whenisolated as described above.

Those of ordinary skill in the art will recognize that triturationthrough reduced bore pipettes is not the only way to isolate spore-likecells from larger, differentiated cells. For example, flow cytometry canalso be used. Alternatively, a suspension containing spore-like cellsand differentiated cells can be passed through a filter having pores ofa particular size. The size of the pores within the filter (and,similarly, the diameter of the pipette used for trituration) can bevaried, depending on how stringent one wishes the isolation procedure tobe. Generally, the smaller the pores within the filter, or the smallerthe diameter of the pipette used for trituration, the fewer the numberof differentiated cells that will survive the isolation procedure.

At the time of isolation, spore-like cells may not express the receptorsor other cellular components that make differentiated cells susceptibleto attack by infectious agents. This is another characteristic that canbe exploited to identify and isolate spore-like cells. For example, onecan infect a tissue or cell culture with an infectious agent and thenseparate the live spore-like cells from the differentiated cells thatwere killed by the infectious agent.

The features and characteristics described above can be used todistinguish spore-like cells from previously identified cell types. Forexample, the spore-like cells of the invention can be identified bytheir ability to differentiate into a variety of terminallydifferentiated cell types found in mature animals (such as thoseillustrated in the Examples below), their typical spherical shape, smallsize (as small as 0.1–0.3 μ in diameter and generally 1.0 to 3.0 μ indiameter), and cytoarchitecture (which includes relatively large amountsof nuclear material, relatively small amounts of cytoplasm, and aglycolipid- or mucopolysaccharide-rich coat), their ability to survivein environments in which other cell types would die (e.g., environmentsin which there is a low, or even non-existent, oxygen supply,environments in which the temperature is higher or lower than othercells can tolerate (without special protective measures), environmentscontaining toxins, non-physiological salt concentrations, acids, bases,or radioenergy that other cells cannot tolerate).

When cultured as described in the Examples below, spore-like cellsproliferate more rapidly and into more types of differentiated cellsthan do terminally differentiated cells or mesenchymal stem cells. Cellviability can be assessed using standard techniques, including visualobservation with light or scanning electron microscopes and Trypan blueexclusion.

Spore-like cells have been isolated from body fluids (e.g., the blood)as well as from solid functional organs such as the liver, but it is notclear that they originate exclusively in either of these places. It maybe that tissues and organs are the primary sources for spore-like cells,which appear in body fluids only secondarily, for example, when thecells are “washed out” of those tissues. However, it is also possiblethat spore-like cells originate in bodily fluids or from the same sourceas other cells that are present in bodily fluids (e.g., spore-like cellsmay originate in the bone marrow). If so, spore-like cells could then besubsequently delivered from those fluids to specific tissues. Moreover,delivery may be upregulated when the tissue is affected by, for example,a disorder, a regenerative process, or wound healing.

Without limiting the invention to spore-like cells that differentiate bya particular mechanism, it is believed that the rate and nature ofspore-like cell differentiation can be influenced by altering the numberand type of mature cells that come into contact (physical or functionalcontact) with spore-like cells. A mature cell is infunctional contactwith a spore-like cell when the mature cell emits a chemical signal(e.g. a growth factor, cytokine, or neurotransmitter) that is detectedby the spore-like cell. For example, when isolating spore-like cellsfrom the liver, the more mature hepatocytes that remain in the cultureof spore-like cells, the more quickly the spore-like cells willdifferentiate and the more likely it is that they will differentiateinto hepatocytes. Thus, it is believed that spore-like cells proliferateand differentiate in response to agents (e.g., growth factors orhormones) within tissue, including tissue that has been injured or thatis otherwise associated with a medical disorder. These agents guidedifferentiation so that the spore-like cells or their progeny come toexpress some or all of the same phenotypic markers expressed by maturecells normally present in the tissue in which they have been placed.Spore-like cells can be influenced by agents within tissues regardlessof their origin (i.e., regardless of whether the spore-like cellsoriginate in the blood, another body fluid, the bone marrow, or a solid,functional tissue or organ).

Spore-like cells can be used to maintain the integrity and function of awide variety of tissues as well as to reengineer, repair, or otherwiseimprove tissue associated with a medical disorder. For example,spore-like cells can be used to maintain or reengineer: bone; bonemarrow; muscle (e.g., smooth, skeletal, or cardiac muscle); connectivetissue (e.g., cartilage, ligaments, tendons, pleura, or fibroustissues); epithelial and mucous membranes; lung tissue; vascular tissue;nervous tissue (e.g., neurons and glial cells in the central orperipheral nervous systems), glandular tissue (e.g., tissue of thethyroid gland, adrenal gland, or sweat or sebaceous glands); epithelialcells, keratinocytes, or other components of the skin; lymph nodes; theimmune system; reproductive organs; or any of the internal organs (e.g.,liver, kidney, pancreas, stomach, bladder, or any portion of thealimentary canal). This list is intended to illustrate, not limit, thetypes of cells and tissues that can benefit from administration ofspore-like cells. For example, life-like artificial skin can be producedby culturing spore-like cells and allowing them, when applied to aliving body or used in conjunction with present skin replacementmethods, to differentiate into epidermal and dermal cells (includingmelanocytes) as well as into hair follicles, sweat glands, sebaceousglands, ganglia, and similar adnexal structures. Those of ordinary skillin the art will recognize many other therapeutic uses for spore-likecells.

Spore-Like Cell Differentiation

Spore-like cells or their progeny can differentiate into a number ofdifferent cell types. For example, spore-like cells can differentiateinto epithelial cells, keratinocytes, melanocytes, adipocytes, myocytes,chondrocytes, osteocytes, alveolar cells, hepatocytes, renal cells,adrenal cells, endothelial cells, islet cells (e.g., alpha cells, deltacells, PP cells, and beta cells), blood cells (e.g., leukocytes,erythrocytes, macrophages, and lymphocytes) retinal cells (and othercells involved in sensory perception, such as those that form hair cellsin the ear or taste buds on the tongue), and fibroblasts or other celltypes present in organs and connective tissues.

Spore-like cells and their progeny can be induced to differentiate in avariety of ways and may or may not be committed to a particulardifferentiation pathway. One method of inducing differentiation is toallow spore-like cells or their progeny to establish contact (e.g.,physical contact) with a solid support. For example, spore-like cellscan differentiate when they establish contact with (e.g., adhere to) aglass or plastic surface, a mesh, or other substrate suitable for use intissue culture or administration to a patient.

Spore-like cells can also differentiate when they establish contact witha tissue within a patient's body or are sufficiently close to a tissueto be influenced by substances (e.g., growth factors, enzymes, orhormones) released from the tissue. Thus, differentiation of aspore-like cell can be influenced by virtue of signals the cell receivesfrom the surrounding tissue. Such signaling would occur, for example,when a receptor on the surface of a spore-like cell, or on the surfaceof a cell descended from a spore-like cell, bound and transduced asignal from a molecule such as a growth factor, enzyme,neurotransmitter, or hormone that was released by a tissue within thepatient.

Alternatively, or in addition, spore-like cells can be induced todifferentiate by adding a substance (e.g., a growth factor, enzyme,hormone, or other signaling molecule) to the cell's environment. Forexample, a substance can be added to a culture dish containingspore-like cells, to a mesh or other substrate suitable for applyingspore-like cells to a tissue, or to a tissue within a patient's body.When a substance that induces spore-like cells to differentiate isadministered, either systemically or locally, it can be administeredaccording to pharmaceutically accepted methods. For example, proteins,polypeptides, or oligonucleotides can be administered in aphysiologically compatible buffer, with or without a carrier orexcipient. Of course, either the cells within a patient's body or thecells being administered (here, spore-like cells or their progeny) canbe made to express particular factors following genetic manipulation.For example, spore-like cells can be made to express hormones, such asinsulin, by transfecting them with gene constructs that includesequences that encode these factors. Thus, spore-like cells or theirprogeny can differentiate either in culture or in a patient's body, andmay do so following contact with a solid support or exposure tosubstances that are either naturally expressed, exogenouslyadministered, or expressed as a result of genetic manipulation.Regardless of the stimulus for differentiation, spore-like cells thathave differentiated, or that will do so, sufficiently to aid in themaintenance or repair of tissue, can be administered to a patient (e.g.,at the site of a burn or other traumatized area of skin, a bonefracture, a torn ligament, an-atrophied muscle, a malfunctioning gland,or an area adversely affected by a neurodegenerative process (or traumaor ischemia, as occurs with a cerebrovascular accident) or autoimmuneresponse). Based on simple observation, it appears that when spore-likecells come into contact with each other, they orchestrate their owndevelopment. It appears that once contact is initiated, a developmentalpattern is set into motion.

Another way to promote proliferation without differentiation is toexpose the spore-like cells, particularly those isolated from the skin,to agonists of Notch function, as described in U.S. Pat. No. 5,780,300.Agonists of Notch include, but are not limited to, proteins such asDelta or Serrate or Jagged (Lindsell et al., Cell 80:909–917, 1995) orbiologically active fragments thereof. These proteins or proteinfragments mediate binding to Notch and thereby activate the Notchpathway. Spore-like cells isolated from the skin can be contacted inculture with agonists of Notch or can be transfected with genes thatencode Notch agonists. As described above, the techniques required totransfect cells in culture are routinely practiced by those of ordinaryskill in the art. Spore-like cells that remain undifferentiated inculture can differentiate when administered to a patient; theirdifferentiation being orchestrated by the microenvironment theyencounter within the patient.

As described in Example 7, below, many cells isolated as spore-likecells from the liver express bile after 12 days in culture. Bileexpression can be seen following staining by Hall's technique usingFouchet's reagent (FIG. 7E). Bile pigments can also be identified by atleast two other standard histological stains, the Gmelin test, andStein's method. Similarly, there are a number of standard assays forglycolipids, which are carbohydrate and lipid compounds that contain 1mole each of a fatty acid, sphingosine, and hexose. Common reactions forcarbohydrates include the periodic acid-Schiff (PAS) reaction, diastase,alcian blue staining, colloidal iron, and hyaluronidase. Spore-likecells isolated from adult liver are stained by PAS and mucicarminestains, which indicates that these cells are coated withmucopolysaccharids and glycolipids.

While spore-like cells or their progeny may eventually become fullydifferentiated, and while this is desirable in some circumstances (e.g.,where the cells are used to essentially recreate a histologically matureand complete tissue), fully differentiated cells are not alwaysnecessary for successful treatment; spore-like cells or their progenyneed only differentiate to a point sufficient to treat the patient. Forexample, spore-like cells used to treat diabetes need not everdifferentiate into cells that are indistinguishable from fullydifferentiated p cells within the islets of Langerhans. To the contrary,spore-like cells or their progeny need only differentiate to the pointwhere they express sufficient insulin to treat the diabetic patient.

Excluded from the invention are cells having characteristics that renderthem indistinguishable from previously identified stem cells (e.g.,mesenchymal stem cells), precursor cells (e.g., the islet cellprecursors described by Cornelius et al. (Horn. Metab. Res. 29:271–277(1997)), or the progenitors from central nervous tissue described byShihabuddin et al. (Exp. Neurol. 148:577–586 (1997)) or Weiss et al. (J.Neurosci. 16:7599–7609 (1996)) or terminally differentiated cells. Thesecharacteristics can be assessed by those of ordinary skill in the art innumerous ways (e.g., by histological, biochemical, or, preferably,electron microscopic analysis).

Methods of Treatment

A. Administration of Spore-like Cells and Their Progeny Via Hydrogel

The novel cell types described herein can be administered to a patientby way of a composition that includes spore-like cells, or theirprogeny, and a liquid hydrogel. This cell-hydrogel mixture can beapplied directly to a tissue that has been damaged. For example, asdescribed in U.S. Ser. No. 08/747,036, a hydrogel-cell mixture cansimply be brushed, dripped, or sprayed onto a desired surface or pouredor otherwise made to fill a desired cavity or device. The hydrogelprovides a thin matrix or scaffold within which the spore-like cellsadhere and grow. These methods of administration may be especially wellsuited when the tissue associated with a patient's disorder has anirregular shape or when the cells are applied at a distant site (e.g.,when spore-like cells are placed beneath the renal capsule to treatdiabetes).

Alternatively, the hydrogel-cell mixture can be introduced into apermeable, biocompatible support structure so that the mixtureessentially fills the support structure and, as it solidifies, assumesthe support structure's shape. Thus, the support structure can guide thedevelopment and shape of the tissue that matures from spore-like cells,or their progeny, that are placed within it. As described further below,the support structure can be provided to a patient either before orafter being filled with the hydrogel-cell mixture. For example, thesupport structure can be placed within a tissue (e.g., a damaged area ofthe skin, the liver, or the skeletal system) and subsequently filledwith the hydrogel-cell composition using a syringe, catheter, or othersuitable device. When desirable, the shape of the support structure canbe made to conform to the shape of the damaged tissue. In the followingsubsections, suitable support structures, hydrogels, and deliverymethods are described (cells suitable for use are described above).

1. Hydrogels

The hydrogels used to practice this invention should be biocompatible,biodegradable, capable of sustaining living cells, and, preferably,capable of solidifying rapidly in vivo (e.g., in about five minutesafter being delivered to the support structure). Large numbers ofspore-like cells can be distributed evenly within a hydrogel; a hydrogelcan support approximately 5×10⁶ cells/ml. Hydrogels also enablediffusion so that nutrients reach the cells and waste products can becarried away.

A variety of different hydrogels can be used to practice the invention.These include, but are not limited to: (1) temperature dependenthydrogels that solidify or set at body temperature (e.g., PLURONICS™);(2) hydrogels cross-linked by ions (e.g., sodium alginate); (3)hydrogels set by exposure to either visible or ultraviolet light, (e.g.,polyethylene glycol polylactic acid copolymers with acrylate endgroups); and (4) hydrogels that are set or solidified upon a change inpH (e.g., TETRONICS™).

Materials that can be used to form these different hydrogels include,but are not limited to, polysaccharides such as alginate,polyphosphazenes, and polyacrylates, which are cross-linked ionically,block copolymers such as PLURONICS™ (also known as POLOXAMERS™), whichare poly(oxyethylene)-poly(oxypropylene) block polymers solidified bychanges in temperature, TETRONICS™ (also known as POLOXAMINES™), whichare poly(oxyethylene)-poly(oxypropylene) block polymers of ethylenediamine solidified by changes in pH.

Ionic Hydrogels

Ionic polysaccharides, such as alginates or chitosan, can also be usedto suspend living cells, including spore-like cells and their progeny.These hydrogels can be produced by cross-linking the anionic salt ofalginic acid, a carbohydrate polymer isolated from seaweed, with ions,such as calcium cations. The strength of the hydrogel increases witheither increasing concentrations of calcium ions or alginate. U.S. Pat.No. 4,352,883 describes the ionic cross-linking of alginate withdivalent cations, in water, at room temperature, to form a hydrogelmatrix.

Spore-like cells are mixed with an alginate solution, the solution isdelivered to an already implanted support structure, whichthen-solidifies in a short time due to the presence of physiologicalconcentrations of calcium ions in vivo. Alternatively, the solution isdelivered to the support structure prior to implantation and solidifiedin an external solution containing calcium ions.

In general, these polymers are at least partially soluble in aqueoussolutions (e.g., water, aqueous alcohol solutions that have charged sidegroups, or monovalent ionic salts thereof). There are many examples ofpolymers with acidic side groups that can be reacted with cations (e.g.,poly(phosphazenes), poly(acrylic acids), and poly(methacrylic acids)).Examples of acidic groups include carboxylic acid groups, sulfonic acidgroups, and halogenated (preferably fluorinated) alcohol groups.Examples of polymers with basic side groups that can react with anionsare poly(vinyl amines), poly(vinyl pyridine), and poly(vinyl imidazole).

Polyphosphazenes are polymers with backbones consisting of nitrogen andphosphorous atoms separated by alternating single and double bonds. Eachphosphorous atom is covalently bonded to two side chains.Polyphosphazenes that can be used have a majority of side chains thatare acidic and capable of forming salt bridges with di- or trivalentcations. Examples of acidic side chains are carboxylic acid groups andsulfonic acid groups.

Bioerodible polyphosphazenes have at least two different types of sidechains: acidic side chains capable of forming salt bridges withmultivalent cations, and side chains that hydrolyze in vivo (e.g.,imidazole groups, amino acid esters, glycerol, and glucosyl).Bioerodible or biodegradable polymers (i.e., polymers that dissolve ordegrade within a period that is acceptable in the desired application(usually in vivo therapy), will degrade in less than about five yearsand most preferably in less than about one year, once exposed to aphysiological solution of pH 6–8 having a temperature of between about25° C. and 38° C. Hydrolysis of the side chain results in erosion of thepolymer. Examples of hydrolyzing side chains are unsubstituted andsubstituted imidizoles and amino acid esters in which the side chain isbonded to the phosphorous atom through an amino linkage.

Methods for synthesis and the analysis of various types ofpolyphosphazenes are described in U.S. Pat. Nos. 4,440,921, 4,495,174,and 4,880,622. Methods for the synthesis of the other polymers describedabove are known to those of ordinary skill in the art. See, for exampleConcise Encyclopedia of Polymer Science and Engineering, J. I.Kroschwitz, Ed., John Wiley and Sons, New York, N.Y., 1990. Manypolymers, such as poly(acrylic acid), alginates, and PLURONICS™ arecommercially available.

Water soluble polymers with charged side groups are cross-linked byreacting the polymer with an aqueous solution containing multivalentions of the opposite charge, either multivalent cations if the polymerhas acidic side groups, or multivalent anions if the polymer has basicside groups. Cations for cross-linking the polymers with acidic sidegroups to form a hydrogel include divalent and trivalent cations such ascopper, calcium, aluminum, magnesium, and strontium. Aqueous solutionsof the salts of these cations are added to the polymers to form soft,highly swollen hydrogels.

Anions for cross-linking the polymers to form a hydrogel includedivalent and trivalent anions such as low molecular weight dicarboxylateions, terepthalate ions, sulfate ions, and carbonate ions. Aqueoussolutions of the salts of these anions are added to the polymers to formsoft, highly swollen hydrogels, as described with respect to cations.

For purposes of preventing the passage of antibodies into the hydrogel,but allowing the entry of nutrients, a useful polymer size in thehydrogel is in the range of between 10 and 18.5 kDa. Smaller polymersresult in gels of higher density with smaller pores.

Temperature-Dependent Hydrogels

Temperature-dependent, or thermosensitive, hydrogels can also be used inthe methods of the invention. These hydrogels have so-called “reversegelation” properties, i.e., they are liquids at or below roomtemperature, and gel when warmed to higher temperatures (e.g., bodytemperature). Thus, these hydrogels can be easily applied at or belowroom temperature as a liquid and automatically form a semi-solid gelwhen warmed to body temperature. As a result, these gels are especiallyuseful when the support structure is first implanted into a patient, andthen filled with the hydrogel-cell composition. Examples of suchtemperature-dependent hydrogels are PLURONICS™ (BASF-Wyandotte), such aspolyoxyethylene-polyoxypropylene F-108, F-68, and F-127, poly(N-isopropylacrylamide), and N-isopropylacrylamide copolymers.

These copolymers can be manipulated by standard techniques to affecttheir physical properties such as porosity, rate of degradation,transition temperature, and degree of rigidity. For example, theaddition of low molecular weight saccharides in the presence and absenceof salts affects the lower critical solution temperature (LCST) oftypical thermosensitive polymers. In addition, when these gels areprepared at concentrations ranging between 5 and 25% (WNV) by dispersionat 4° C., the. viscosity and the gel-sol transition temperature areaffected, the gel-sol transition temperature being inversely related tothe concentration. These gels have diffusion characteristics capable ofallowing spore-like cells and their progeny to survive and be nourished.

U.S. Pat. No. 4,188,373 describes using PLURONIC™ polyols in aqueouscompositions to provide thermal gelling aqueous systems. U.S. Pat. Nos.4,474,751, '752, '753, and 4,478,822 describe drug delivery systems thatutilize thermosetting polyoxyalkylene gels. With these systems, both thegel transition temperature and/or the rigidity of the gel can bemodified by adjustment of the pH and/or the ionic strength, as well asby the concentration of the polymer.

pH-Dependent Hydrogels

Other hydrogels suitable for use in the methods of the invention arepH-dependent. These hydrogels are liquids at, below, or above specificpH values, and gel when exposed to specific pHs, for example, 7.35 to7.45, the normal pH range of extracellular fluids within the human body.Thus, these hydrogels can be easily delivered to an implanted supportstructure as a liquid and automatically form a semi-solid gel whenexposed to body pH. Examples of such pH-dependent hydrogels areTETRONICS™ (BASF-Wyandotte) polyoxyethylene-polyoxypropylene polymers ofethylene diamine, poly(diethyl aminoethyl methacrylate-g-ethyleneglycol), and poly(2-hydroxymethyl methacrylate). These copolymers can bemanipulated by standard techniques to affect their physical properties.

Light Solidified Hydrogels

Other hydrogels that can be used to administer spore-like cells or theirprogeny are solidified by either visible or ultraviolet light. Thesehydrogels are made of macromers including a water soluble region, abiodegradable region, and at least two polymerizable regions (see, e.g.,U.S. Pat. No. 5,410,016). For example, the hydrogel can begin with abiodegradable, polymerizable macromer including a core, an extension oneach end of the core, and an end cap on each extension. The core is ahydrophilic polymer, the extensions are biodegradable polymers, and theend caps are oligomers capable of cross-linking the macromers uponexposure to visible or ultraviolet light, for example, long wavelengthultraviolet light.

Examples of such light solidified hydrogels include polyethylene oxideblock copolymers, polyethylene glycol polylactic acid copolymers withacrylate end groups, and 10K polyethylene glycol-glycolide copolymercapped by an acrylate at both ends. As with the PLURONIC™ hydrogels, thecopolymers comprising these hydrogels can be manipulated by standardtechniques to modify their physical properties such as rate ofdegradation, differences in crystallinity, and degree of rigidity.

Thus, a variety of hydrogels can be used to practice the presentinvention. They include, but are not limited to: (1) temperaturedependent hydrogels that solidify or set at body temperature, e.g.,PLURONICS™; (2) hydrogels cross-linked by ions, e.g., sodium alginate;(3) hydrogels set by exposure to either visible or ultraviolet light,e.g., polyethylene glycol polylactic acid copolymers with acrylate endgroups; and (4) hydrogels that are set or solidified upon a change inpH, e.g., TETRONICS™.

The materials that can be used to form these various hydrogels includepolysaccharides such as alginate, polyphosphazenes, and polyacrylates,which are cross-linked ionically, or block copolymers such as PLURONICS™(also known as POLOXAMERS™), which arepoly(oxyethylene)-poly(oxypropylene) block polymers solidified bychanges in temperature, or TETRONICS™ (also known as POLOXAMINES™),which are poly(oxyethylene)-poly(oxypropylene) block polymers ofethylene diamine solidified by changes in pH.

2. Preparation of Hydrogel-Cell Mixtures

Once a hydrogel of choice (e.g., a thermosensitive polymer at between 5and 25% (W/V), or an ionic hydrogel such as alginate dissolved in anaqueous solution (e.g., a 0.1 M potassium phosphate solution, atphysiological pH, to a concentration between 0.5% to 2% by weight) isprepared, isolated spore-like cells or their progeny are suspended inthe polymer solution. If desired, the concentration of the cells canmimic that of the tissue to be generated. For example, the concentrationof cells can range between 10 and 100 million cells/ml (e.g., between 20and 50 million cells/ml or between 50 and 80 million cells/ml). Ofcourse, the optimal concentration of cells to be delivered into thesupport structure may be determined on a case by case basis, and mayvary depending on cell type and the region of the patient's body intowhich the support structure is implanted or onto which it is applied. Tooptimize the procedure (i.e., to provide optimal viscosity and cellnumber), one need only vary the concentrations of the cells or thehydrogel.

3. Support Structures

The support structure is a permeable structure having pore-like cavitiesor interstices that shape and support the hydrogel-cell mixture. Forexample, the support structure can be a porous polymer mesh, or anatural or synthetic sponge. The porosity of the support structureshould be such that nutrients can diffuse into the structure, therebyeffectively reaching the cells inside, and waste products produced bythe cells can diffuse out of the structure.

The support structure can be shaped to conform to the space in which newtissue is desired. For example, the support structure can be shaped toconform to the shape of an area of the skin that has been burned or theportion of cartilage or bone that has been lost. Depending on thematerial from which it is made, the support structure can be shaped bycutting, molding, casting, or any other method that produces a desiredshape (as described below, in some instances, the support structure canbe shaped by hand). Moreover, the shaping process can occur eitherbefore or after the support structure is filled with the hydrogel-cellmixture. For example, a support structure can be filled with ahydrogel-cell mixture and, as the hydrogel hardens, molded into adesired shape by hand.

As the hydrogel solidifies, it will adopt the flexibility and resiliencyof the support structure, which is important for accommodation ofcompressive and tensile forces. Thus, for example, replaced skin couldaccommodate tensile forces associated with pulling and stretching, aswell as compressive forces associated with weight bearing, as occurs,for example, on the soles of the feet. The flexibility and resiliency ofthe support structure also provides greater ease of administration. Forexample, in many currently available skin replacement methods, thetissue is extremely delicate and must be handled with the utmost care.

The support structure is also biocompatible (i.e., it is not toxic tothe spore-like cells suspended therein) and can be biodegradable. Thus,the support structure can be formed from a synthetic polymer such as apolyanhydride, polyorthoester, or polyglycolic acid. The polymer shouldprovide the support structure with an adequate shape and promote cellgrowth and proliferation by allowing nutrients to reach the cells bydiffusion. Additional factors, such as growth factors, other factorsthat induce differentiation or dedifferentiation, secretion products,immunomodulators, anti-inflammatory agents, regression factors,biologically active compounds that promote innervation or enhance thelymphatic network, and drugs, can be incorporated into the polymersupport structure.

An example of a suitable polymer is polyglactin, which is a 90:10copolymer of glycolide and lactide, and is manufactured as VICRYL™braided absorbable suture (Ethicon Co., Somerville, N.J.). Polymerfibers (such as VICRYL™), can be woven or compressed into a felt-likepolymer sheet, which can then be cut into any desired shape.Alternatively, the polymer fibers can be compressed together in a moldthat casts them into the shape desired for the support structure. Insome cases, additional polymer can be added to the polymer fibers asthey are molded to revise or impart additional structure to the fibermesh.

For example, a polylactic acid solution can be added to this sheet ofpolyglycolic fiber mesh, and the combination can be molded together toform a porous support structure. The polylactic acid binds thecrosslinks of the polyglycolic acid fibers, thereby coating theseindividual fibers and fixing the shape of the molded fibers. Thepolylactic acid also fills in the spaces between the fibers. Thus,porosity can be varied according to the amount of polylactic acidintroduced into the support. The pressure required to mold the fibermesh into a desirable shape can be quite moderate. All that is requiredis that the fibers are held in place long enough for the binding andcoating action of polylactic acid to take effect.

Alternatively, or in addition, the support structure can include othertypes of polymer fibers or polymer structures produced by techniquesknown in the art. For example, thin polymer films can be obtained byevaporating solvent from a polymer solution. These films can be castinto a desired shaped if the polymer solution is evaporated from a moldhaving the relief pattern of the desired shape. Polymer gels can also bemolded into thin, permeable polymer structures using compression moldingtechniques known in the art.

Many other types of support structures are also possible. For example,the support structure can be formed from sponges, foams, corals, orbiocompatible inorganic structures having internal pores, or mesh sheetsof interwoven polymer fibers. These support structures can be preparedusing known methods.

4. Application of the Support Structure

Any of the liquid hydrogel-cell mixtures described above can be placedin any of the permeable support structures (also described above). FIG.13 is a schematic of a filled support structure in cross-section. Thisstructure is suitable for application of spore-like cells or theirprogeny to the skin. The support structure 10 is formed from a bilayeredmesh of interwoven polymer fibers 12 having epidermal layer 12 a anddermal layer 12 b. The spaces between the fibers form interconnectedpores 14 that are filled with liquid hydrogel-cell mixture. Within ashort time of placing the mixture in the support structure (e.g., inapproximately three to five minutes), hydrogel 16 solidifies, therebykeeping the suspended cells 18 within the pores 14 of support structure10. The solidified hydrogel 16 helps maintain the viability of the cellsby allowing diffusion of nutrients (including growth and differentiationfactors) and waste products through the interconnected pores of thesupport structure. The ultimate result being the growth of new skin andits engraftment to the patient's body.

The liquid hydrogel-cell mixture can be delivered to the shaped supportstructure either before or after the support structure is implanted inor applied to a patient. The specific method of delivery will depend onwhether the support structure is sufficiently “sponge-like” for thegiven viscosity of the hydrogel-cell composition, i.e., whether thesupport structure easily retains the liquid hydrogel-cell mixture beforeit solidifies. Sponge-like support structures can be immersed within,and saturated with, the liquid hydrogel-cell mixture, and subsequentlyremoved from the mixture. The hydrogel is then allowed to solidifywithin the support structure. The hydrogel-cell-containing supportstructure is then implanted in or otherwise administered to the patient.

The support structure can also be applied to the patient before thehydrogel completely solidifies. Alternatively, a sponge-like supportstructure can be injected with the liquid hydrogel-cell mixture, eitherbefore or after the support structure is implanted in or otherwiseadministered to the patient. The hydrogel-cell mixture is then allowedto solidify.

The volume of the liquid hydrogel-cell mixture injected into the supportstructure is typically less than, but somewhat comparable to, the volumeof the support structure, i.e., the volume of the desired tissue to begrown.

Support structures that do not easily retain the liquid compositionrequire somewhat different methods. In those cases, for example, thesupport structure is immersed within and saturated with the liquidhydrogel-cell mixture, which is then allowed to partially solidify. Oncethe cell-containing hydrogel has solidified to the point where thesupport structure can retain the hydrogel, the support structure isremoved from the partially solidified hydrogel, and, if necessary,partially solidified hydrogel that remains attached to the outside ofthe support structure is removed (e.g., scraped off the structure).

Alternatively, the liquid hydrogel-cell mixture can be delivered into amold containing the support structure. For example, the liquidhydrogel-cell mixture can be injected into an otherwise fluid-tight moldthat contains the support structure and matches its outer shape andsize. The hydrogel is then solidified within the mold, for example, byheating, cooling, light-exposure, or pH adjustment, after which, thehydrogel-cell-containing support structure can be removed from the moldin a form that is ready for administration to a patient.

In other embodiments, the support structure is implanted in or otherwiseadministered to the patient (e.g., placed over the site of a burn orother wound, placed beneath the renal capsule, or within a region of thebody damaged by ischemia), and the liquid hydrogel-cell mixture is thendelivered to the support structure. The hydrogel-cell mixture can bedelivered to the support using any simple device, such as a syringe orcatheter, or merely by pouring or brushing a liquid gel onto a supportstructure (e.g., a sheet-like structure).

Here again, the volume of hydrogel-cell composition added to the supportstructure should approximate the size of the support structure (i.e.,the volume displaced by the desired tissue to be grown). The supportstructure provides space and a structural template for the injectedliquid hydrogel-cell mixture. As described above, some of thehydrogel-cell mixture may leak from the support structure prior tosolidifying. However, in this event, existing tissue beneath orsurrounding the support structure would sufficiently constrain theliquid hydrogel-cell mixture until it gels.

In any of the above cases, the hydrogel is solidified using a methodthat corresponds to the particular hydrogel used (e.g., gently heating acomposition including a PLURONIC™ temperature-sensitive hydrogel).

To apply or implant the support structure, the implantation site withinthe patient can be prepared (e.g., in the event the support structure isapplied to the skin, the area can be prepared by debridement), and thesupport structure can be implanted or otherwise applied directly at thatsite. If necessary, during implantation, the site can be cleared ofbodily fluids such as blood (e.g., with a burst of air or suction).

EXAMPLES

The present invention will be further understood by reference to thefollowing non-limiting examples.

Example 1

Spore-like cells were isolated from human blood as follows. Five cc's ofwhole blood were acquired from an adult human and placed in a tubecontaining an anti-coagulant. The blood sample was then centrifuged at1200 rpm for approximately five minutes. The supernatant was removed,and the resulting pellet was resuspended in 15 cc's of DMEM/F-12 mediumsupplemented with a combination of the following hormones and nutrients:glucose (23 mM), transferrin (10 mg/ml), insulin (20 mg/ml), putricine(10 mM), selenium (100 nM), progesterone (10 nM) (Life Technologies,Baltimore, Md.), EGF (20 ng/ml), and bFGF (20 ng/ml) (CollaborativeBiomedical Products, Chicago, Ill.). The resulting suspension wastransferred to 75 cm² tissue culture flasks and incubated in 5% CO₂ at37° C. The media were changed every 3–4 days. Cells were passaged every7–9 days. Initially, these culture flasks appeared to contain manyhematopoeitic cells (e.g., red blood cells), but over time (usually, amatter of several days), these cells disappeared, leaving onlyspore-like cells. After several days in culture, the spore-like cellsmultiplied and coalesced to form clusters of cells. Trypan blueexclusion revealed cell viability to be greater than 90%. FIGS. 11 and12A are photographs of cultures that include undifferentiated spore-likecells isolated from adult human blood. The cells shown in FIG. 12A wereisolated seven days earlier and are viewed with phase contrastmicroscopy. Immunofluorescent staining was then performed. At this time,some of the cells expressed nestin (see FIG. 12B).

Example 2

Spore-like cells were isolated from the skin of an adult rodent asfollows. Excisional biopsies of the skin of adult Fisher rats were madeunder sterile conditions. The biopsied tissue, which included the dermisand epidermis, was placed in a petri dish containing cold phosphatebuffered saline (PBS) and antibiotics (penicillin (50 mU/ml) andstreptomycin (90 mg/ml)). The epidermis was scraped with, a #11 scalpelto disassociate epidermal cells, and the tissue was then transferred toa second petri dish (also containing cold PBS and antibiotics) where thedermis was scraped with a #11 scalpel. The cells that were dissociatedwere then centrifuged at 1200 rpm (GLC-2B, Sorvall, Wilmington, Del.)for five minutes and resuspended in 10 ml of 0.05% trypsin (LifeTechnologies, Baltimore, Md.). Following resuspension in trypsin, thetissue was incubated at 37° C. for five minutes. Ten ml of Dulbecco'sModified Eagle Medium (DMEM)/F-12 containing 10% heat inactivated fetalbovine serum (FBS) (Life Technologies, Baltimore, Md.) was added todeactivate the trypsin.

The tissue was then triturated, first with a normal bore Pasteur pipetteand subsequently with a series of fire polished pipettes having boresreduced to about 15 μm. The number of pipettes required can varydepending upon how frequently they become clogged with tissue.Trituration was carried out until the tissue was dispersed as a finesuspension. The suspension was then centrifuged at 1200 rpm (GLC-2B,Sorvall, Wilmington, Del.) for five minutes. The supernatant was removedand the pellet was resuspended in 15 ml of DMEM/F-12 medium supplementedwith a hormone mixture containing glucose (23 mM), transferrin (10mg/ml), insulin (20 mg/ml), putricine (10 mM), selenium (100 nM),progesterone (10 nM) (Life Technologies, Baltimore, Md.), EGF (20 ng/ml)and bFGF (20 ng/ml) (Collaborative Biomedical Products, Chicago, Ill.).The suspension was transferred to 75 cm² tissue culture flasks(Collaborative Biomedical Products, Chicago, Ill.) and incubated at 37°C. in 5% CO₂. The media was changed every three days, and the cells werepassaged every 7–9 days. The cells that attached to the tissue cultureflask appeared to differentiate more readily.

Spore-like cells isolated from the skin will differentiate upon exposureto the processes and basal nutrient media described in U.S. Pat. No.5,292,655. Alternatively, growth factors that cause spore-like cells tomitose (e.g., epidermal growth factor (EGF), basic fibroblast growthfactor (bFGF) and other cytokines) can be applied to help maintain thecells in an undifferentiated state. For example, the isolated cells canbe cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplementedwith a hormone mixture containing glucose, transferrin, insulin,putricine, selenium, progesterone, EGF, and bFGF.

Spore-like cells were also isolated from excisional biopsies of the skinof adult pigs according to the same protocol described here for theadult rat.

Example 3

Spore-like cells were isolated from adult rat heart according to theprotocol described in Example 2. The newly isolated cells, which areshown in FIG. 3A, include undifferentiated spore-like cells. After threedays in culture, early myocardial cells can be seen (FIG. 3B), and aftertwo weeks in culture, Purkinje-like structures can be seen (FIG. 3C).

Example 4

Spore-like cells were isolated from adult rat intestine according to theprotocol described in Example 2. The newly isolated cells, as shown inFIG. 4A, include undifferentiated spore-like cells. After three days inculture, clusters of small intestinal cells (FIG. 4B) and autonomicneurons (FIG. 4C) can be seen.

Example 5

Spore-like cells were isolated from an adult rat bladder according tothe protocol described in Example 2. The newly isolated cells, which areshown in FIG. 5A, include undifferentiated spore-like cells. After twodays in culture, the isolated spore-like cells, or their progeny, appearto be differentiating into mature bladder cells (FIG. 5B).

Example 6

Spore-like cells were isolated from an adult rat kidney according to theprotocol described in Example 2. Cells newly isolated from the kidney ofan adult rat, which are shown in FIG. 6A, include undifferentiatedspore-like cells. After three days in culture, aggregates of cellsresembling kidney structures can be seen (FIG. 6B).

Example 7

Spore-like cells were isolated from an adult rat liver according to theprotocol described in Example 2. Because the liver is highlyvascularized, the intact tissue was washed with PBS. Cells newlyisolated from the liver of an adult rat, which are shown in FIGS. 7A and7C, include undifferentiated spore-like cells. After three days inculture, an aggregate of cells resembling a differentiating liverstructure can be seen (FIG. 7B). After seven days in culture, cellsresembling hepatocytes can be seen (FIG. 7D).

Example 8

Spore-like cells were isolated from adult mammalian lungs according tothe protocol described in Example 2. Spore-like cells were isolated fromthe lungs of adult rats (see FIGS. 8A–8C) and sheep (see FIG. 8D). Thenewly isolated cells shown in FIG. 8A include undifferentiatedspore-like cells. After six weeks in culture, alveolar-like cells can beseen (FIGS. 8B and 8C). After 30 days in culture, spore-like cellsisolated from an adult sheep have formed alveolar-like structures (FIG.8D) similar to those seen in the lungs of adult cats (FIG. 8E;Histology, F. Hammersen, Ed., Urban & Schwarzenberg, Baltimore-Munich,1980, FIG. 321).

Example 9

Spore-like cells were isolated from adult rat adrenal glands accordingto the protocol described in Example 2. Undifferentiated spore-likecells isolated from the adrenal gland of an adult rat can be seen at Day0 in FIGS. 9A and 9B (see the arrows). After two days in culture,primitive adrenal cells can be seen (FIGS. 9C and 9D).

Example 10

Spore-like cells were isolated from the pancreas of an adult human andfrom the pancreas of an adult rat. The dissections were carried out in10% cold fetal serum albumin according to the protocol described inExample 2. Significantly, spore-like cells have been isolated from aportion of the rat pancreas that remained after the islets were removedby ductal injection of collagenase (as described, for example, by Suttonet al., Transplantation, 42:689–691, 1986).

Islet-like structures that formed in cultures of spore-like cellsisolated from islet-free pancreatic tissue are shown in FIGS. 10A–10C.After six days in culture, more than 100 islet-like structures werepresent per field (see FIGS. 10A and 10B), even though the spore-likecells first placed in culture were isolated from a tissue from which theislets had been removed. When the islet-like structures thatnevertheless developed were immunostained, insulin expression can beseen (FIG. 10C).

Example 11

Due in part to the unusual appearance of spore-like cells under thelight microscope, the cells were examined under an electron microscope.Scanning and electron microscopy was performed according to standardprotocols. The electron micrographs revealed several interestingfeatures. For example, the range of spore-like cell sizes may be greaterthan first appreciated with the light microscope. Some of the spore-likecells shown in FIG. 1A have a diameter of approximately 0.3 microns. Theunique cytoarchitecture of the spore-like cell is apparent when viewedwith transmission electron microscopy (see FIGS. 2A–2D) or followingnuclear staining (such as the 4′6-diamidino-2-phenylindole (DAPI) staindescribed in Example 12). The interior of the cell is consumed largelywith diffuse nuclear material and the cell is surrounded by a “zebra”coating, which is associated with deposits of glycolipids (i.e.,carbohydrate and fat). For example, zebra bodies (so-called because oftheir striped appearance) are associated with mucopolysaccharidoses,such as Hurler's syndrome or with Fabry's disease, in which glycolipidsaccumulate due to an enzyme deficiency. Spore-like cells thus appear,during at least one stage of their existence, to be unique packets ofDNA.

Example 12

A massive accumulation of nuclear material is also apparent whenspore-like cells are stained for nucleic acids by methods known to thoseof ordinary skill in the art. For example, DNA can be stained witheither 4′6,-diamidino-2-phenylindole (DAPI) for total DNA staining orwith propidium iodide for staining of double-stranded DNA and RNA. DAPIand propidium iodide can be added directly to anti-fade mounting medium(e.g., 90% glycerol, 1× PBS, and 2.5% 1,4-diazabicyclo[2,2,2]octane(DABCO) (Sigma Chemical Co., St. Louis, Mo.). Spore-like cells stainedwith DAPI contained a great deal of nuclear material; the ratio ofnuclear to cytoplasmic material was much higher in spore-like cells thanone would expect in most fully differentiated cell types.

Example 13

Four tissues (lung, liver, fascia, and spinal cord) were obtained fromthree animals (Fisher rats) and kept in cold storage for five days. Morespecifically, each tissue type was removed from an animal less than twohours after the animals was killed and placed in a 50 cc centrifuge tube(Fisher Scientific, Pittsburg, Pa.) filled with PBS. The tubes werestored at 4° C. without supplemental oxygen for five days. Spore-likecells were then isolated as follows. After excision from the animal, andusing sterile technique, the selected tissue was placed in cold PBScontaining penicillin (50 mU/ml) and streptomycin (90 mg/ml) (Gibco,Grand Island, N.Y.). The tissue was then manually disassociated with a#11 scalpel, and the disassociated cells were collected bycentrifugation at 1200 rpm for five minutes. The tissue was thenresuspended in ten ml of 0.05% trypsin (w/v) for five minutes at 37° C.The trypsin was inactivated by adding 10 ml of DMEM/F-12 medium (Gibco)supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco).The cells were then dispersed by trituration using progressivelynarrower fire-polished, reduced-bore pasteur pipettes. While theaperatures are not measured, the opening of the smallest-bore pipettewas approximately 15 μm. The dispersed cells were collected bycentrifugation at 1200 rpm for five minutes. The resulting pellet wasresuspended in 10 ml of DMEM/F-12 medium containing 33 mM glucose (SigmaChemical Co., St. Louis, Mo.), 10 mg/ml transferrin (Sigma), 20 mg/mlinsulin (Sigma), 10 mM putrescine (Sigma), 100 nM selenium (Sigma), 10nM progesterone (Sigma), 20 ng/ml EGF (Peprotech, Rocky Hill, N.J.), and20 ng/ml bFGF (Collaborative Biomedical, Raynham, Mass.). The primarycell suspension was incubated at 37° C. in 5% CO₂, and the media werechanged every 3 days. Cells were passaged every 7–9 days by collectingthe nonadherent cell aggregates, centrifuging them at 1200 rpm for fiveminutes and removing the media. Cells were resuspended in fresh media,triturated using narrow fire polished reduced bore pasteur pipettes. Thecell suspension was then divided into two suspensions and placed intotwo new culture dishes.

The technique described above was slightly modified to isolate hepatictissue: hepatic tissue was washed with cold PBS prior to disassociation.

Standard hematoxalin and eosin (H&E) staining was performed on tissuefixed with 10% formalin. A simple Hall's stain was performed onliver-derived spore-like cells for the presence of bile. Standard stainsfor mucicarmine and periodic acid-Schiff were also performed.

To assess cellular proliferation, the time that was required for apopulation of cells to double its number was estimated using periodicphase microscopy field counts (10 fields counted and averaged at 100×)or viable cell counts using trypan blue with a hemocytometer.

Based on the exclusion of trypan blue, approximately 50% of thespore-like cells within each of the four tissues that were exposed to 4°C., without supplemental oxygen, for five days, remained viable at theend of that period. Moreover, the spore-like cells isolated from lung,liver, fascia, and spinal cord retained their ability to proliferate anddifferentiate into tissue-specific structures.

Example 14

After being killed, whole animals (Fisher rats) were placed in plasticbags and stored in a freezer at −86° C. After being frozen for eithertwo or eight weeks, the animals were removed from the freezer and placedin a 37° C. water bath until their tissues thawed. Four tissues (lung,liver, fascia, and spinal cord) were then harvested, and spore-likecells were isolated as described in Example 13 and assessed by trypanblue exclusion for viability. Spore-like cells could be obtained fromoxygen-deprived and deeply frozen tissue just as they were fromoxygen-deprived and chilled tissue. Approximately 50% of the spore-likecells within each of the four tissues that were exposed to −86° C.,without supplemental oxygen, for two or eight weeks, remained viable atthe end of those periods. Moreover, the spore-like cells isolated fromlung, liver, fascia, and spinal cord retained their ability toproliferate and differentiate into tissue-specific structures.

Example 15

Four tissues (lung, liver, fascia, and spinal cord) were obtained fromthree animals (Fisher rats) and heated to 85° C. for 30 minutes. Morespecifically, each tissue type was removed from an animal less than twohours after the animals was killed and placed in a 50 cc centrifuge tube(Fisher Scientific, Pittsburg, Pa.) filled with PBS. The tubes were thenplaced in a heated water bath as the temperature of the bath was raisedto 85° C. The temperature was monitored with sterile thermometers, whichwere placed within each tube. The tubes were left in the water bath for45 minutes after the temperature reached 85° C. The tissue was thenallowed to cool to room temperature, and spore-like cells were isolatedas described in Example 13.

Based on the exclusion of trypan blue, approximately 50% of thespore-like cells within each of the four tissues that were heated to 85°C. for 30 minutes remained viable at the end of that period. Moreover,the spore-like cells isolated from lung, liver, fascia, and spinal cordretained their ability to proliferate and differentiate intotissue-specific structures.

Example 16

Spore-like cells isolated from the central nervous system fail to showactivity in a microculture tetrazolium assay (conducted as described inMarshall et al. (Growth Regulation 5:69–84, 1995)), but do so when theydifferentiate into cells with more usual and recognizablecharacteristics. This assay tests for redox activity and, when negative,indicates that no cells are present. This assay therefore providesfurther evidence that spore-like cells are unique from known cell typesand that they exist in a dormant state without apparent metabolicactivity.

Example 17

Blood from a patient who developed type I diabetes eight years earlierwas collected in a standard tube with anticoagulant and subsequentlyfrozen at −85° C. without preparation (i.e., without treatment with acryopreservative or other substance) for up to 26 weeks. Spore-likecells were successfully isolated from this blood sample after up to fourfreeze-thaw cycles. No other intact cells were observed (as was expectedas no known cell types are known to survive these conditions).

More specifically, after each thaw cycle, one cc of blood was added to20 ccs of complete medium (DMEM/F12 with progesterone, b-FGF, and EGF)After the cells were added, the medium was triturated with a pasteurpipette and reduced-bore pasteur pipettes and filtered through, first, a100 micron filter and then a 40 micron filter. The cells were thenplaced in a 175 cm² flask and incubated at 37° C. with 5% CO₂.Initially, a few intact spore-like cells were suspended in the mediawith gel-like properties, with 3–5 cc of fresh media added every 3 to 5days. The media then assumed a liquid quality, with rapid spore-likecell replication (ten doublings in 5 days). At about day 12, thespore-like cells formed small clusters. At this point, 0.5 cc of 30%glucose was added to the culture flask, raising the concentration ofglucose from about 100 mg/dl to 500 mg/dl (a hyperglycemic condition).In response, the clusters enlarged, and frequent nucleated cells couldbe seen within 5 days. Insulin production was demonstrated by bothstandard immunofluorescent assays and by RT-PCR for insulin mRNA.

OTHER EMBODIMENTS

One of ordinary skill in the art will appreciate that the spore-likecells described herein can be administered in connection with existingtissue engineering methods, in lieu of differentiated cells incell-based therapies, and in lieu of cells presently administeredfollowing genetic manipulation.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, that the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims.

Other aspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for isolating a spore-like cell population from a mammalianbiological tissue or cell containing fluid, the method comprising (a)obtaining a mammalian tissue or cell containing fluid and exposing themammalian tissue or cell containing fluid to an environment in whichdifferentiated or partially differentiated cells in the tissue or fluiddie, wherein the environment includes one or more conditions selectedfrom the group consisting of temperature of 42° C. or greater, freezing,non-physiological salt concentration, essential absence of oxygen for atleast four hours, size separation and passaging in cell culture,treatment with acid or base, radiation and drying, and (b) separatingthe population of viable spore-like cells from the dead differentiatedor partially differentiated cells.
 2. The method of claim 1, furthercomprising disrupting the tissue or fluid either before or after step(a) and separating the viable spore-like cell from the deaddifferentiated or partially differentiated cells by size separation. 3.The method of claim 1 wherein the spore-like cell population fails todemonstrate activity in a microtetrazolium assay.
 4. The method of claim1 wherein the spore-like cells contain between approximately 50 and 90%by volume nuclear material.
 5. The method of claim 1 wherein thespore-like cells have a diameter of approximately 15 microns or less. 6.The method of claim 1, wherein the spore-like cells have a diameter ofbetween 0.1 and 3.0 microns.
 7. The method of claim 1 wherein the tissueor fluid is treated with salt, acid or base and the spore-like cellsisolated.
 8. The method of claim 1, wherein the biological tissuecomprises a tissue that originates from the endoderm.
 9. The method ofclaim 1, wherein the biological tissue comprises a tissue thatoriginates from the mesoderm.
 10. The method of claim 1, wherein thebiological tissue comprises a tissue that originates from the ectoderm.11. The method of claim 1, wherein the biological fluid comprises blood,urine, or saliva.
 12. The method of claim 1, wherein the biologicalfluid is cerebrospinal fluid.
 13. The method of claim 1, wherein theenvironment is an oxygen-poor environment.
 14. The method of claim 1,wherein the environment is one in which the temperature is above orbelow the range of temperatures in which differentiated or partiallydifferentiated cells can survive.
 15. The method of claim 1, wherein theenvironment contains a toxin or infectious agent that killsdifferentiated or partially differentiated cells.
 16. The method ofclaim 1 wherein the environment contains radiation or is dessicating.17. The method of claim 1 further comprising placing the cell populationinto a matrix for implantation into a site for tissue repair,augmentation or regeneration.
 18. The method of claim 17, furthercomprising implanting the matrix into a site for tissue repair,augmentation or regeneration.
 19. The method of claim 1 furthercomprising culturing the spore-like cell population.
 20. The method ofclaim 18 further comprising implanting the matrix into a tissue selectedfrom the group consisting of the visual system, auditory system, nasalepithelium, alimentary canal, pancreas, gallbladder, bladder, kidney,liver, heart, respiratory system, nervous system, reproductive system,endocrine system, immune system, bone, muscle, tooth, nail, and skin.21. The method of claim 17 wherein the matrix is a hydrogel.
 22. Themethod of claim 1 wherein the tissue is selected from the groupconsisting of cardiac, smooth and skeletal muscle, intestine, bladder,kidney, liver, lung, adrenal gland, skin, retina, nasal epithelium,brain, spinal cord, periosteum, perichondrium, fascia, and pancreas. 23.The method of claim 1 wherein the spore-like cell population is frozenafter isolation.
 24. The method of claim 1 further comprising inducingthe isolated spore-like cells in the population to differentiate. 25.The method of claim 21 wherein the spore-like cells in the populationare introduced into a support structure.
 26. The method of claim 17wherein the matrix is a porous polymer mesh, suture, film or sponge. 27.The method of claim 1, wherein the biological fluid is cerebrospinalfluid.